Penn Researchers Develop New Test To Detect Rare
Proteins in BloodApplications of New Method Will Make Possible Earliest Warning
of Cancer and Other Diseases

(Philadelphia, PA) - Researchers at the University of Pennsylvania
School of Medicine have developed a paradigm-shifting method
for detecting small amounts of proteins in the blood. Applications of
this method will make discerning low-abundance molecules associated with
cancers (such as breast cancer), Alzheimer’s disease, prion diseases,
and possibly psychiatric diseases relatively easy and more accurate compared
with the current methodology, including the widely used ELISA (enzyme-linked
immunoadsorbent assay).

ELISA is a common immune-system-based assay that uses enzymes linked to
an antibody or antigen as a marker for picking out specific proteins.
For example, it is used as a diagnostic test to determine exposure to
infectious agents, such as HIV, by identifying antibodies present in a
blood sample.

The sensitivity of detecting molecules by the new method, called FACTT,
short for Fluorescent Amplification Catalyzed by T7-polymerase Technique,
is five orders of magnitude (100,000 times) greater than that of ELISA,
the Penn researchers found.

“The current ELISA tests can only detect proteins when they are
in high abundance,” says Zhang. “But the problem is that many
of the functional proteins - those that have a role in determining your
health - exist in very low amounts until diseases are apparent and cannot
be detected or measured at early stages of medical pathology. It was important
to develop a technique that can detect these rare molecules to detect
abnormalities at an early stage.”

The FACTT technology uses a different enzyme amplification system so quantitative
signals can be obtained from even a few protein molecules compared to
ELISA. “The technology is remarkably adaptable to any protein and
can be performed in an automated format,” notes Greene. He states
that the technology will soon be robotized so as to be able to screen
for many rare disease-causing proteins using tiny amounts of blood. “It
is even possible that one could screen for multiple diseases at the same
time and produce a precise accounting of whether disease-causing molecules
are present at an early time when disease can be readily treated,”
adds Greene.

Greene also noted that the FACTT technology represents the further evolution
of an earlier approach that was developed in collaboration with Professor
of Pharmacology James Eberwine, PhD, also from Penn.
The earlier technique employed radioisotopes.

Development of a test for the cancer marker Her2/neu
The researchers compared detection of Her2/neu in the blood between ELISA
and FACTT. Her2/neu proteins were in fact first identified by the Greene
laboratory in the early 1980s, and the Her2/neu gene was found by other
scientists to be overexpressed in breast cancer. Her2/neu is normally
a low-abundance molecule that becomes overexpressed in more than 30 percent
of primary breast, ovarian, and pancreatic tumors. Part of the Her2/neu
molecule is shed from the surface of tumor cells and has been detected
in the blood of breast-cancer patients. Higher blood concentrations of
Her2/neu correlate with a lower response rate to chemotherapy and shorter
survival time after relapse.

The Greene lab developed mouse models that carry cancer cells overexpressing
Her2/neu. When these cells are implanted into animals they form tumors
exactly like breast tumors in humans. Using ELISA, the researchers could
not detect Her2/neu from mouse blood until the tumors reached an inoperable
size, but with the new FACTT technology they could detect Her2/Neu in
some mice when tumors were barely visible and within two days of implantation.
These results indicate that it is possible to detect tumors at very early
stages so that tumor emergence or reoccurrence can be rapidly treated
or even prevented.

Greene’s laboratory established many of the principles of targeted
therapy for Her2/neu tumors and the prototype antibodies that led to the
development of Herceptin, a similar antibody molecule that was created
by Genentech. The Greene laboratory also previously showed that early
treatment of Her2/neu tumors with targeted monoclonal antibodies in animal
models led to far more significant prevention of tumor growth as well
as tumor emergence and reoccurrence.

Greene stresses that early treatment is far more effective than treating
advanced tumors with the same antibodies. Recent clinical trials support
the notion that early treatment prevents tumor reoccurrence in women with
breast tumors. FACTT technology represents a way to couple early diagnosis
with early treatment to prevent tumor emergence.

Detecting Her2/neu in humans for breast cancer
The most widely used clinical Her2/neu tests are IHC (immunohistochemistry)
and FISH (fluorescence in situ hybridization). However, both FISH and
IHC are complex, time-consuming tests.

Patients who test positive for Her2/neu using FISH or IHC have responsive
rates of about 35 percent to the cancer drug Herceptin. Monitoring Her2/neu
status from the blood with a powerful technology such as FACTT represents
an alternative approach compared to IHC or FISH, say the researchers.

Pre-treatment Her2/neu levels correlate with tumor size and the extent
of disease. Post-treatment Her2/neu levels predict disease-specific survival.
A more sensitive assay could more accurately allow treatment of humans
with breast cancer and allow treatment more quickly if the tumor reoccurs.

The researchers collected blood samples from healthy women and breast
cancer patients who did or did not overexpress Her2/neu, as detected by
IHC and FISH. When using the new FACTT method her2/neu positive cancer
patients showed dramatically elevated Her2/neu levels (average: 384 ng/ml),
while the level in Her2/neu-negative breast cancer patients (19.5 ng/ml)
were close to the levels of the healthy control participants (16.6 ng/ml).

Using FACTT, nine out of 10 of the Her2/neu positive patients had elevated
Her2/neu levels and one out of four in the Her2/neu negative group had
elevated Her2/neu levels. Using ELISA only two out of 10 in the Her2/neu
positive group showed elevated Her2/neu levels.

“Clearly the sensitivity of the ELISA assay does not satisfy the
current need for the clinical detection of marker proteins that determine
whether a patient has breast cancer or not,” says Greene.

The researchers have also tried the FACTT method on other rare, but medically
important molecules, such as the prion protein (for mad cow disease with
Mansun Sy at Case Western University) and TNF-alpha (for autoimmune diseases),
and will be developing tests for other cancer markers including lung cancer
and colon cancer. All proteins tested so far with FACTT have been detected
with an over 1000-fold higher sensitivity compared to current technologies.

The researchers say this points to FACTT’s broad applicability and
compatibility with current high-throughput testing technology. This, in
turn, will facilitate the detection of rare markers and not-so-rare targets
from much smaller sample volumes, as well as aid in monitoring marker
levels at much earlier stages of disease.

“The importance of FACTT is that we can still get an accurate description
of the number of molecules that cause disease even when other assays cannot,”
says Greene. The researchers surmise that FACTT could be used to monitor
levels of Her2/neu in already-diagnosed breast cancer patients to monitor
recurrence or treatment effectiveness.

“The critical issue arises when women are diagnosed with early breast
cancer,” adds Greene. “They often have a lumpectomy and are
sometimes treated with radiation or chemotherapy, but despite this conventional
therapy the cancer still can occasionally reoccur,” says Greene.
Detection of very early recurrence is important and Greene feels the power
of this technology will facilitate recognizing early phases of tumor emergence.

Rational targeted therapy has shown in animal models - over 10 years ago
- and more recently in clinical trials that treatment of small or incipient
tumors is a way to prevent tumor emergence or reoccurrence. “Prevention
of the consequences of recurrence is critical since treating advanced
tumors is very complex and difficult,” concludes Greene.

This research was funded in part by The Abramson Family Cancer Research
Institute.

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